The onset and completion of cell division (mitosis) are controlled by activation and inactivation of the cdc2 protein kinase. Molecular analyses have shown that cdc2 kinase activity is regulated in a complex manner involving multiple phosphorylations and the physical association of cdc2 with mitotic cyclins, proteins which oscillate in abundance over the course of the cell cycle. It is generally thought that inactivation of the cdc2 kinase directly results from the destruction of mitotic cyclins. To understand the pathways controlling cdc2 kinase activation and inactivation at the cellular level, we have developed an approach that combines techniques of biochemistry and cell physiology using permeabilized cells and cell-free assays. We have shown that physiological levels of Ca2+ can inactivate the cdc2 kinase in the absence of cyclin destruction, suggesting the hypothesis that a Ca2+-dependent mechanism initiates inactivation, and that the destruction of cyclin serves to ensure that another round of kinase activation does not take place before another round of DNA replication. In many cells, the activation of protein kinase C (PKC) leads to a reduction of intracellular Ca2+ levels. We have shown that the activation of PKC blocks cdc2 kinase inactivation without blocking the destruction of mitotic cyclins. This finding further demonstrates that cdc2 kinase inactivation and cyclin destruction can be uncoupled, and supports the view that intracellular Ca2+ levels are a key component of the pathway controlling exit from mitosis.